We have examined the role of activator/enhancer elements in the regulation of eukaryotic viral gene expression. In SV40, a pair of 72 bp tandem repeat sequences, located approximately 100-200 bp from the site of initiation of transcription, are required for transcription. These sequences activate other genes placed in their immediate vicinity. Similar sequences have been identified in the long terminal repeat (LTR) of murine sarcoma virus (MSV) and have been found capable of replacing the 72 bp repeat sequences of SV40 to form a viable virus (SVrMSV). In this study, employing an assay for viral T-antigen expression as well as an in vitro assay for early gene expression involving the prokaryotic gene, chloramphenicol acetyltransferase (CAT), host-specific gene activation was examined. Using identical promoter regions from SV40, the SV40 tandem repeats were found to be more active in monkey kidney cells, whereas the MSV repeats were more active in mouse cells. Therefore, at least some activators appear to function in a host-specific manner. In addition, the MSV enhancer has been shown to work both 5 feet and 3 feet to the SV40 promoter in the CAT assay although not at equivalent rates. The two MSV repeats are not identical in sequence and have been found to enhance expression at different rates.